# US 8,771,694 B2 · Immunoconjugates and humanized antibodies specific for B-cell lymphoma and leukemia cells

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- Number: US 8,771,694 B2
- Document ID: US8771694B2
- Status: Granted
- Kind: B2
- Application number: 09/988,013
- Filed: 2001-11-16
- Published: 2003-06-05
- Granted: 2014-07-08
- Earliest claimed: 1994-08-12
- Assignees: Immunomedics, Inc.
- Applicants: Shui-on Leung; Hans Hansen
- Inventors: Shui-on Leung; Hans Hansen
- Web page: https://patentgrep.com/patent/US8771694B2

## CPC

- C07K 16/2803: {against the immunoglobulin superfamily}
- A61K 2039/505: {comprising antibodies}
- C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
- C07K 2317/41: Glycosylation, sialylation, or fucosylation
- C07K 2317/77: Internalization into the cell
- C07K 2319/00: Fusion polypeptide
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)

## Abstract

A chimeric LL2 monoclonal antibody (mAb) is described in which the variable regions of the murine LL2 mAb are recombinantly joined to the human constant region domains, which retains the immunospecificity and B-cell lymphoma and leukemia cell internalization capacity of the murine LL2 (mAB), and which exhibits reduced human anti-mouse antibody production activity (HAMA). A humanized LL2 mAb is described in which the CDRs are recombinantly joined to a framework sequence of human variable regions, and subsequently linked to human constant region domains, which retains the immunospecificity and B-lymphoma and leukemia cell internalization capacities of the murine and chimeric LL2 mABs, and has the potential for exhibiting reduced HAMA. Isolated DNAs encoding the LL2 variable and CDR framework regions are described. Conjugates of chimeric and humanized chimeric LL2 antibodies with cytotoxic agents or labels find use in therapy and diagnosis of B-cell lymphomas and leukemias.

## Claims

### Claim 1

A method of designing amino acid sequences of variable domains of a humanized monoclonal antibody comprising:
(a) comparing the amino acid sequences of the light and heavy chain variable domains of a monoclonal antibody to be humanized with the amino acid sequences of the light and heavy chain variable domains of two or more human antibodies;
(b) selecting framework regions from a first human antibody for the light chain and from second and third human antibodies for the heavy chain based on the sequence comparison, wherein the heavy chain FR1, FR2 and FR3 are selected from the second human antibody and FR4 is selected from the third human antibody;
(c) incorporating the framework regions selected in step (b) with the corresponding light and heavy chain complementarity determining regions of the monoclonal antibody to be humanized, to design humanized light and heavy chain variable domain amino acid sequences;
(d) preparing DNA sequences encoding the humanized light and heavy chain variable domain amino acid sequences;
(e) operably incorporating the variable domain DNA sequences into at least one vector comprising DNA sequences encoding the constant domains of the human light and heavy chain regions;
(f) introducing the at least one vector into a cell; and
(g) culturing the cell containing the at least one vector under conditions to produce the humanized monoclonal antibody
wherein the heavy chain FR4 is selected from the human NEWM antibody, the light chain framework regions are selected from the human REI antibody, and the heavy chain FR1,
FR2 and FR3 are selected from the human EU antibody.

### Claim 2 (depends on 1)

The method according to claim 1, further comprising retaining selected amino acid residues from the framework regions of the monoclonal antibody to be humanized in the corresponding framework regions of the humanized variable domains where said selected amino acids are predicted to have contacts with said complementarity determining regions.

### Claim 3 (depends on 2)

The method according to claim 2, wherein said selected amino acid residues are within a 4.5 Angstrom radius of any atoms within a complementarity determining regions of the light or heavy chain of the humanized monoclonal antibody.